Samuel Santos-Ribeiro

Trends in ectopic pregnancy rates following assisted reproductive technologies in the UK: a 12-year nationwide analysis including 160 000 pregnancies

STUDY QUESTION Have the advancement of assisted reproductive technologies (ART) and changes in the incidence of specific causes of infertility-altered ectopic pregnancy (EP) rates following ART over time in the UK? SUMMARY ANSWER EP rates in the UK following IVF/ICSI have progressively decreased, and this appears to be associated with a reduction in the incidence of tubal factor infertility and the increased use of both a lower number of embryos transferred and extended embryo culture. WHAT IS KNOWN ALREADY Historically, EP rates following ART are known to have increased over time. However, the impact of progress in ART procedures and changes in both policy and the incidence of specific causes of infertility on the overall EP rate in the UK has yet to be studied. STUDY DESIGN, SIZE, DURATION A population-based retrospective analysis was carried out on all pregnancies following ART cycles carried out in the UK between 2000 and 2012 included in the anonymized database of the Human Fertilisation and Embryology Authority. PARTICIPANTS/MATERIALS, SETTING, METHODS Overall, 161 967 treatment cycles resulting in a pregnancy were included in the analysis. Among them, 8852 pregnancies occurred after intrauterine insemination (IUI) and 153 115 following IVF/ICSI. MAIN RESULTS AND THE ROLE OF CHANCE During this period of 12 years, ∼1.4% (n = 2244) of all pregnancies following ART were an EP. Crude EP rates were significantly higher after IVF/ICSI when compared with following IUI (1.4 versus 1.1%, P = 0.043). The incidence of EP decreased significantly over time for IVF/ICSI cycles [incidence rate ratios (IRR) 0.96 per year, 95% confidence interval (CI) 0.94-0.97], but not after IUI (IRR 0.96 per year, 95% CI 0.91-1.03).Among pregnancies resulting from IVF/ICSI, multivariable logistic regression analysis demonstrated that the major risk factor for EP was the presence of tubal infertility [adjusted odds ratio (aOR) 2.23, 95% CI 1.93-2.58), followed by the increased number of embryos transferred (aOR 1.29 for 2 versus 1 embryo transferred, 95% CI 1.11-1.49; aOR 1.69 for 3 or more versus 1 embryo transferred, 95% CI 1.35-2.11). The use of extended embryo culture to Days 3-4 or 5-7 significantly reduced the risk of EP, when compared with the transfer of early cleavage (Days 1-2) embryos (respectively, aOR 0.85, 95% CI 0.76-0.94; and aOR 0.73, 95% CI 0.63-0.84). Finally, frozen embryo transfer (ET) had no effect on the risk of EP following IVF/ICSI (aOR 0.92, 95% CI 0.76-1.11). LIMITATIONS, REASONS FOR CAUTION Owing to the use of this particular registry data, well-established risk factors of EP, such as smoking habits or uterine surgery, could not be assessed. WIDER IMPLICATIONS OF THE FINDINGS Our results provide the first evidence of a potential benefit-in terms of the reduction in EP rates-of the implementation of national programmes aiming to reduce the incidence of tubal infertility, such as the National Chlamydia Screening Programme. In addition, campaigns for the widespread introduction of single ET may not only reduce the incidence of multiple pregnancies but also the incidence of EP following IVF/ICSI. STUDY FUNDING/COMPETING INTERESTS No funding was obtained for this study, and there are no conflicts of interest to declare.

Ovarian hyperstimulation syndrome after gonadotropin-releasing hormone agonist triggering and "freeze-all": in-depth analysis of genetic predisposition.

PurposeWe report on the results of the whole-genome analysis performed in a patient who developed severe ovarian hyperstimulation syndrome (OHSS) following gonadotropin-releasing hormone (GnRH) agonist triggering in a “freeze-all” protocol.MethodsA 30-year-old patient with polycystic ovary syndrome who developed severe early-onset OHSS with clinical ascites, and slight renal and hepatic dysfunction was admitted for monitoring and treatment with cabergoline and intravenous albumin. Exome sequencing to assess for any known genetic predisposition for OHSS was performed.ResultsNo known genetic variants associated with OHSS predisposition were found.ConclusionsCase reports of severe OHSS following a “freeze-all” strategy are starting to arise, showing that OHSS has not been completely eliminated with this approach. Further studies should be conducted to confirm if such cases may be due to genetic predisposition or not.

Vitamin D deficiency and pregnancy rates following frozen–thawed embryo transfer: a prospective cohort study

STUDY QUESTION What is the effect of vitamin D deficiency on the pregnancy rates following frozen embryo transfer (FET)?. SUMMARY ANSWER Vitamin D deficiency does not affect pregnancy rates in FET cycles. WHAT IS KNOWN ALREADY Although there is evidence that the potential impact of vitamin D deficiency on reproductive outcome may be mediated through a detrimental effect on oocyte or embryo quality, the rationale of our design was based on evidence derived from basic science, suggesting that vitamin D may have a key role in endometrial receptivity and implantation. Only few retrospective clinical studies have been published to date with conflicting results. STUDY DESIGN, SIZE, DURATION This study is the first prospective observational cohort study from the Centre for Reproductive Medicine at the University Hospital of Brussels. The duration of the study was 1 year. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 280 consecutive patients, who had at least one blastocyst frozen and were planned for a FET, were enrolled in the study following detailed information and signing of a written informed consent. Serum analysis of 25-OH vitamin D was measured on the day of embryo transfer, and the impact of vitamin deficiency was investigated on reproductive outcomes. MAIN RESULTS AND THE ROLE OF CHANCE Among all patients, 45.3% (n = 127) had vitamin D deficiency (<20 ng/ml), and 54.6% (n = 153) had vitamin D levels ≥20 ng/ml. Positive human chorionic gonadotrophin rates were similar among patients with vitamin D deficiency and women with total serum 25-OH vitamin D levels ≥20 ng/ml (40.9 versus 48.3%, P = 0.2). Similarly, no difference was found in clinical pregnancy rates in women with vitamin D deficiency [32.2% (41/127)] compared with those with higher vitamin D levels [37.9% (58/153)]; P = 0.3. When analyzing the results according to different thresholds, as proposed by the Endocrine Society, clinical pregnancy rates were comparable between vitamin D deficient (<20 ng/ml), vitamin D insufficient (20-30 ng/ml) and vitamin D replete women (≥30 ng/ml) [32.3% (41/127) versus 39.5% (36/91) versus 35.5% (22/62), respectively, P = 0.54]. Multivariate logistic regression analysis showed that vitamin D status is not related to pregnancy outcome. LIMITATIONS, REASONS FOR CAUTION Ethnicity in relation to vitamin D status was not assessed, given that the vast majority of patients included in our study were Caucasian, whereas we did only assess 25-OH vitamin D levels and not bioavailable vitamin D. Furthermore, although we failed to find a difference between vitamin D deficient women and women with vitamin D levels ≥20 ng/ml, we need to underscore that our study was powered to detect a difference of 15% in clinical pregnancy rates. WIDER IMPLICATIONS OF THE FINDINGS Vitamin D deficiency does not significantly impair pregnancy rates among infertile women undergoing frozen-thawed cycles. The measurement of vitamin D levels in this population should not be routinely recommended. STUDY FUNDING/COMPETING INTERESTS No external funding was used for this study. No conflicts of interest are declared.

Should we continue to measure endometrial thickness in modern-day medicine? The effect on live birth rates and birth weight

The evaluation of endometrial thickness (EMT) is still part of standard cycle monitoring during IVF, despite the lack of robust evidence of any value of this measurement to predict little revalidation in contemporary medical practice; other tools, however, such as endocrine profile monitoring, have become increasingly popular. The aim of this study was to reassess whether EMT affects the outcome of a fresh embryo transfer in modern-day medicine, using a retrospective, single-centre cohort of 3350 IVF cycles (2827 women) carried out between 2010 and 2014. In the multivariate regression analysis, EMT was non-linearly associated with live birth, with live birth rates being the lowest with an EMT less than 7.0 mm (21.6%; P < 0.001) and then between 7.0 mm and 9.0 mm (30.2%; P = 0.008). An EMT less than 7.0 mm was also associated with a decrease in neonatal birthweight z-scores (-0.40; 95% CI -0.69 to -0.12). In conclusion, these results reaffirm the use of EMT as a potential prognostic tool for live birth rates and neonatal birthweight in contemporary IVF, namely when considered together with other ovarian stimulation monitoring methods, such as the late-follicular endocrine profile.

Impact of late-follicular phase elevated serum progesterone on cumulative live birth rates: is there a deleterious effect on embryo quality?

STUDY QUESTION Is elevated late-follicular phase progesterone (EP) associated with a deleterious impact on embryo quality (EQ) and cumulative live birth rates (LBRs)? SUMMARY ANSWER EP was associated with a decrease in embryo utilization and cumulative LBRs. WHAT IS KNOWN ALREADY Ovarian stimulation promotes the production of progesterone (P) which adversely affects IVF pregnancy outcomes. However, evidence regarding a potential association between EP an EQ is lacking. STUDY DESIGN, SIZE, DURATION A retrospective analysis of all GnRH antagonist down-regulated ICSI cycles followed by a fresh embryo transfer (ET) between 2010 and 2015 was performed. The sample was stratified according to the following P levels on the day of ovulation triggering: ≤0.50, 0.51-1.49 and ≥1.50 ng/ml. The primary outcomes were embryo utilization rates (number of embryos transferred or cryopreserved) and cumulative LBR, defined as the occurrence of the first live-birth after either the fresh or one of the subsequent frozen ET. PARTICIPANTS/MATERIALS, SETTING, METHODS Overall, 3400 cycles were included in the analysis, using multivariable regression to account for potential confounding. MAIN RESULTS AND THE ROLE OF CHANCE Female age and the number of oocytes retrieved increased significantly with increasing serum P values. Utilization rates decreased linearly as P increased for Day 3 embryos (72.3, 63.0 and 45.4%, respectively), while for Day 5 embryos only the EP group was associated with a significant decrease (48.8, 47.8 and 38.8%, respectively). EP was also associated with decreased fresh and cumulative LBRs. LIMITATIONS REASONS FOR CAUTION The main limitations of this study were its retrospective nature and the fact that it was restricted to GnRH antagonist cycles. WIDER IMPLICATIONS OF THE FINDINGS These results raise the question whether EP may also be associated with a decrease in cumulative pregnancy outcomes by increasing embryo wastage. Further studies may evaluate the potential benefit of additional measures besides the freeze-all strategy to avoid this issue, such as lowering the stimulation dose or applying a step-down protocol. STUDY FUNDING/COMPETING INTEREST(S) None.

Frozen embryo transfer: a review on the optimal endometrial preparation and timing

STUDY QUESTION What is the optimal endometrial preparation protocol for a frozen embryo transfer (FET)? SUMMARY ANSWER Although the optimal endometrial preparation protocol for FET needs further research and is yet to be determined, we propose a standardized timing strategy based on the current available evidence which could assist in the harmonization and comparability of clinic practice and future trials. WHAT IS KNOWN ALREADY Amid a continuous increase in the number of FET cycles, determining the optimal endometrial preparation protocol has become paramount to maximize ART success. In current daily practice, different FET preparation methods and timing strategies are used. STUDY DESIGN, SIZE, DURATION This is a review of the current literature on FET preparation methods, with special attention to the timing of the embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS Literature on the topic was retrieved in PubMed and references from relevant articles were investigated until June 2017. MAIN RESULTS AND THE ROLE OF CHANCE The number of high quality randomized controlled trials (RCTs) is scarce and, hence, the evidence for the best protocol for FET is poor. Future research should compare both the pregnancy and neonatal outcomes between HRT and true natural cycle (NC) FET. In terms of embryo transfer timing, we propose to start progesterone intake on the theoretical day of oocyte retrieval in HRT and to perform blastocyst transfer at hCG + 7 or LH + 6 in modified or true NC, respectively. LIMITATIONS REASONS FOR CAUTION As only a few high quality RCTs on the optimal preparation for FET are available in the existing literature, no definitive conclusion for benefit of one protocol over the other can be drawn so far. WIDER IMPLICATIONS OF THE FINDINGS Caution when using HRT for FET is warranted since the rate of early pregnancy loss is alarmingly high in some reports. STUDY FUNDING/COMPETING INTEREST(S) S.M. is funded by the Research Fund of Flanders (FWO). H.T. and C.B. report grants from Merck, Goodlife, Besins and Abbott during the conduct of the study. TRIAL REGISTRATION NUMBER Not applicable.

Cyclin E1 plays a key role in balancing between totipotency and differentiation in human embryonic cells

STUDY HYPOTHESIS We aimed to investigate if Cyclin E1 (CCNE1) plays a role in human embryogenesis, in particular during the early developmental stages characterized by a short cell cycle. STUDY FINDING CCNE1 is expressed in plenipotent human embryonic cells and plays a critical role during hESC derivation via the naïve state and, potentially, normal embryo development. WHAT IS KNOWN ALREADY A short cell cycle due to a truncated G1 phase has been associated with the high developmental capacity of embryonic cells. CCNE1 is a critical G1/S transition regulator. CCNE1 overexpression can cause shortening of the cell cycle and it is constitutively expressed in mouse embryonic stem cells and cancer cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS We investigated expression of CCNE1 in human preimplantation embryo development and embryonic stem cells (hESC). Functional studies included CCNE1 overexpression in hESC and CCNE1 downregulation in the outgrowths formed by plated human blastocysts. Analysis was performed by immunocytochemistry and quantitative real-time PCR. Mann-Whitney statistical test was applied. MAIN RESULTS AND THE ROLE OF CHANCE The CCNE1 protein was ubiquitously and constitutively expressed in the plenipotent cells of the embryo from the 4-cell stage up to and including the full blastocyst. During blastocyst expansion, CCNE1 was downregulated in the trophectoderm (TE) cells. CCNE1 shortly co-localized with NANOG in the inner cell mass (ICM) of expanding blastocysts, mimicking the situation in naïve hESC. In the ICM of expanded blastocysts, which corresponds with primed hESC, CCNE1 defined a subpopulation of cells different from NANOG/POU5F1-expressing pluripotent epiblast (EPI) cells and GATA4/SOX17-expressing primitive endoderm (PrE) cells. This CCNE1-positive cell population was associated with visceral endoderm based on transthyretin expression and marked the third cell lineage within the ICM, besides EPI and PrE, which had never been described before. We also investigated the role of CCNE1 by plating expanded blastocysts for hESC derivation. As a result, all the cells including TE cells re-gained CCNE1 and, consequently, NANOG expression, resembling the phenotype of naïve hESC. The inhibition of CCNE1 expression with siRNA blocked proliferation and caused degeneration of those plated cells. LIMITATIONS, REASONS FOR CAUTION The study is based on a limited number of good-quality human embryos donated to research. WIDER IMPLICATIONS OF THE FINDINGS Our study sheds light on the processes underlying the high developmental potential of early human embryonic cells. The CCNE1-positive plenipotent cell type corresponds with a phenotype that enables early human embryos to recover after fragmentation, cryodamage or (single cell) biopsy on day 3 for preimplantation genetic diagnosis. Knowledge on the expression and function of genes responsible for this flexibility will help us to better understand the undifferentiated state in stem cell biology and might enable us to improve technologies in assisted reproduction. LARGE SCALE DATA NA STUDY FUNDING AND COMPETING INTERESTS: This research is supported by grants from the Fund for Scientific Research - Flanders (FWO-Vlaanderen), the Methusalem (METH) of the VUB and Scientific Research Fond Willy Gepts of UZ Brussel. There are no competing interests.

Birthweight of singletons born after cleavage-stage or blastocyst transfer in fresh and warming cycles

STUDY QUESTION Does extended culture to the blastocyst stage affect singleton birthweight after either fresh or vitrified-warmed embryo transfer? SUMMARY ANSWER Singleton birthweight z-scores did not vary significantly after a fresh blastocyst transfer, whereas the additional effect of vitrification remains inconclusive. WHAT IS KNOWN ALREADY Observational studies have associated extended culture with an increased risk of preterm birth and low birthweight. On the contrary, in terms of birthweight and gestational age, singletons born after vitrification have been associated with a better perinatal outcome when compared to those born following a fresh transfer. STUDY DESIGN, SIZE, DURATION Our post-hoc cohort analysis on neonatal outcomes included 447 liveborn singletons was derived from a recent retrospective analysis on cumulative live birth rates after cleavage-stage and blastocyst transfers. These babies were born following a fresh single cleavage-stage transfer (FCT Day 3, n = 113), fresh single blastocyst transfer (FBT Day 5, n = 218), vitrified-warmed cleavage-stage transfer (VCT Day 3, n = 58) or vitrified-warmed blastocyst transfer (VBT Day 5, n = 58). PARTICIPANTS/MATERIALS, SETTING, METHODS Singleton birthweight was the primary outcome measure. Gestational age and gender of the newborn were accounted for by using birthweight z-scores in a multivariable linear regression analysis, adjusting for other confounders (maternal age, BMI, parity and smoking behaviour). Vanishing twins were excluded from the analysis. MAIN RESULTS AND THE ROLE OF CHANCE A significantly lower z-score was observed after blastocyst transfer compared to cleavage-stage transfer in the vitrified-warmed Day 5 group (P = 0.013), a difference not observed in the fresh transfer groups (P = 0.32). Following multivariable regression analysis [adjusted regression coefficient (95% confidence interval)], the FCT and FBT groups showed no significant influence on the birthweight z-scores after fresh transfer [-0.19 (-0.44; 0.05)], but the transfer of vitrified blastocysts (VBT) was associated with a lower birthweight [-0.52 (-0.90; -0.15)] compared with the transfer of vitrified cleavage-stage embryos (VCT). LIMITATIONS, REASONS FOR CAUTION The present cohort was relatively small, especially in the vitrified-warmed subgroups. Pregnancy-associated factors possibly influencing birthweight (such as diabetes, hypertension, pre-eclampsia) were also not accounted for in the analysis. WIDER IMPLICATIONS OF THE FINDINGS Different ART procedures, including extended culture and vitrification, may hold potential safety issues. These results require further confirmation in future larger studies. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER N/A.

Vitrified-warmed blastocyst transfer on the 5th or 7th day of progesterone supplementation in an artificial cycle: a randomised controlled trial

Abstract Prospective studies comparing different durations of progesterone supplementation before transfer of vitrified-warmed blastocysts in an artificial cycle are lacking. However, in oocyte donation programmes, the sporadic available evidence demonstrates considerable differences in clinical pregnancy rates according to the duration of progesterone administration. This randomised controlled trial (RCT), included 303 patients undergoing a frozen-thawed embryo transfer (FET) of one or two vitrified-warmed blastocyst(s) in an artificial cycle. Randomisation was performed when the endometrial thickness reached ≥7 mm after oestrogen supplementation. One hundred and fifty two patients in group A received 7 d of vaginal micronised progesterone tablets and 151 patients in group B received 5 d of micronised vaginal progesterone before FET. No differences were seen in clinical pregnancy rate between both groups: 42/152 (27.6%) in group A versus 49/151 (32.5%) in group B. Although no statistically significant difference was observed in clinical pregnancy rates, our study was powered to detect an absolute difference of 16%. In this regard, we cannot exclude that smaller, clinically relevant differences might exist and our study did not have the power to detect this. Patients were also not blinded for the intervention, causing a potential bias.

Cumulative live birth rates after IVF in patients with polycystic ovaries: phenotype matters

RESEARCH QUESTION Do cumulative live birth rates (CLBR) vary among women with different polycystic ovary syndrome (PCOS) phenotypes who undergo IVF/intracytoplasmic sperm injection (ICSI) treatment? DESIGN In this retrospective cohort study, data from 567 patients undergoing an assisted reproductive technology (ART) cycle between January 2010 and December 2015 were collected. Demographical traits, cycle characteristics and clinical and laboratory data were analysed. RESULTS After conventional ovarian stimulation using a gonadotrophin-releasing hormone antagonist protocol, the median number of oocytes retrieved ranged between 11 and 13.5 and did not differ significantly among the studied groups. Live birth rate (LBR) after fresh embryo transfer and CLBR after transfer of all fresh and vitrified embryos were significantly lower in women with hyperandrogenic PCOS phenotypes A (LBR 16.7%, CLBR 25.8%) and C (LBR 18.5%, CLBR 27.8%) compared with women with normoandrogenic PCOS phenotype D (LBR 33.7%, CLBR 48%) (P-value for LBR 0.01 and 0.03, respectively; P-value for CLBR 0.002 and 0.01, respectively) and controls with a polycystic ovarian morphology (LBR 37.1%, CLBR 53.3%) (P-value for LBR 0.002 and 0.01, respectively; P-value for CLBR <0.001 and 0.001, respectively). Multivariate regression analysis indicated that after adjustment for relevant confounders, PCOS phenotype was an independent predictor for CLBR. CONCLUSIONS Hyperandrogenic PCOS phenotypes confer significantly lower CLBR compared with their normoandrogenic counterparts. These findings may imply the need for adapted counselling and tailored approaches when treating PCOS patients with hyperandrogenism who require ART.